Biosensoren für die mikrobielle Stammentwicklung im Hochdurchsatzformat

Schendzielorz, Georg; Binder, Stephan; Marienhagen, Jan

doi:10.1007/s12268-014-0429-y

Cited by 2 publications

(5 citation statements)

References 7 publications

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“…The potential to use bioreporters for high‐throughput screening explains the increased interest in these systems (Jeong et al ., ; Ganesh et al ., ; Mahr and Frunzke, ; Schallmey et al ., ). For instance, various mutagenesis techniques lead to large numbers of altered production strains, but without a high‐throughput screening method, only a limited number of variants can be analysed (Binder et al ., ; Schallmey et al ., ; Schendzielorz et al ., ). In the search for novel biocatalysts, screening large metagenomic or biocatalyst mutant libraries can be complicated and time‐consuming without a high‐throughput screening method, although in this field smart and focused libraries are emerging as well (Goldsmith and Tawfik, ; Illanes et al ., ).…”

Section: Introductionmentioning

confidence: 97%

“…Research aiming at the development of whole‐cell bioreporters for a wide range of applications has increased substantially over the last few decades. Applications include detection of pollutants (Reed et al ., ; Cho et al ., ; Webster et al ., ), the search for novel biocatalysts (Choi et al ., ; Jha et al ., ; Siedler et al ., ) and the improvement of strains for the industrial production of small molecules (Mahr and Frunzke, ; Schendzielorz et al ., ; Siedler et al ., ). A whole‐cell bioreporter (hereafter referred to as bioreporter) is a living microorganism containing a sensor molecule that upon binding of a small molecule of interest switches on a reporter, resulting in a detectable phenotype (Hynninen and Virta, ; van der Meer and Belkin, ; Merulla et al ., ).…”

Section: Introductionmentioning

confidence: 98%

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A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

Rossum

Muras

Baur

et al. 2017

Microbial Biotechnology

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SummaryThe use of bioreporters in high‐throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l‐arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l‐arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts.

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Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 98%

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

Rossum

Muras

Baur

et al. 2017

Microbial Biotechnology

View full text Add to dashboard Cite

show abstract

“…Research aiming at the development of whole-cell bioreporters for a wide range of applications has increased substantially over the last few decades. Applications include detection of pollutants [107][108][109] , the search for novel biocatalysts [110][111][112] , and the improvement of strains for the industrial production of small molecules [113][114][115] . A whole-cell bioreporter (hereafter referred to as bioreporter) is a living microorganism containing a sensor molecule that upon binding of a small molecule of interest switches on a reporter, resulting in a detectable phenotype [116][117][118] .…”

Section: Introductionmentioning

confidence: 99%

“…The potential to use bioreporters for high-throughput screening, explains the increased interest in these systems 113,[119][120][121] . For instance, various mutagenesis techniques lead to large numbers of altered production strains, but without a highthroughput screening method, only a limited number of variants can be analyzed 114,121,122 . In the search for novel biocatalysts, screening large metagenomic or biocatalyst mutant libraries, can be complicated and time-consuming without a high-throughput screening method, although in this field smart and focused libraries are emerging as well 39,46 .…”

Section: Introductionmentioning

confidence: 99%

“…Compared to riboswitch-based sensors, the output of transcriptional regulator-based sensors has a higher fold change upon addition of the small molecule and they are applicable for a more diverse range of small molecules due to their more diverse chemistry, consisting of amino acids versus nucleotides54,179 . Therefore transcriptional regulator-based bioreporters are employed here.Transcriptional regulator-based bioreporters are developed for various types of small molecules, including metals180 , amino acids114 , organic acids87 , phenolic compounds110 . In most cases, the small molecules are very small (< 200 Da), although there are a few examples with slightly bigger molecules such as quercetin (302.24 Da)115 or NADPH (744.42 Da) 112 .…”

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confidence: 99%

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Development of a transcriptional regulator-based bioreporter : towards a generic selection method for novel enzymes

Rossum¹

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The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future. Reporter-based screening and selection of enzymes

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Biosensoren für die mikrobielle Stammentwicklung im Hochdurchsatzformat

Cited by 2 publications

References 7 publications

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

Development of a transcriptional regulator-based bioreporter : towards a generic selection method for novel enzymes

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